Download Detection of the reaction intermediates catalyzed by a copper amine

diffraction structural biology
Journal of
Synchrotron
Radiation
Detection of the reaction intermediates catalyzed
by a copper amine oxidase
ISSN 0909-0495
Received 1 May 2010
Accepted 31 August 2010
Misumi Kataoka,a,b‡2Hiroko Oya,a Ayuko Tominaga,a Masayuki Otsu,a
Toshihide Okajima,c Katsuyuki Tanizawac and Hiroshi Yamaguchia,b*
a
School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda,
Hyogo 669-1337, Japan, bPhysical Science Center for Biomolecular Systems Research, Kwansei
Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan, and cThe Institute of Scientific and
Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
E-mail: [email protected]
To reveal the chemical changes and geometry changes of active-site residues
that cooperate with a reaction is important for understanding the functional
mechanism of proteins. Consecutive temporal analyses of enzyme structures
have been performed during reactions to clarify structure-based reaction
mechanisms. Phenylethylamine oxidase from Arthrobacter globiformis (AGAO)
contains a copper ion and topaquinone (TPQox). The catalytic reaction of
AGAO catalyzes oxidative deaminations of phenylethylamine and consists of
reductive and oxidative half-reactions. In the reduction step, TPQox reacts with
a phenylethylamine (PEA) substrate giving rise to a topasemiquinone (TPQsq)
formed Schiff-base and produces phenylacetaldehyde. To elucidate the
mechanism of the reductive half-reaction, an attempt was made to trap the
reaction intermediates in order to analyze their structures. The reaction
proceeded within the crystals when AGAO crystals were soaked in a PEA
solution and freeze-trapped in liquid nitrogen. The reaction stage of each crystal
was confirmed by single-crystal microspectrometry, before X-ray diffraction
measurements were made of four reaction intermediates. The structure at
15 min after the onset of the reaction was analyzed at atomic resolution, and
it was shown that TPQox and some residues in the substrate channel were
alternated via catalytic reductive half-reactions.
Keywords: copper amine oxidase; AGAO; topaquinone; reaction intermediate;
single-crystal microspectroscopy; X-ray crystal structure analysis.
1. Introduction
To elucidate the mechanism of an enzyme reaction, it is
important to characterize the enzymatic properties and then
determine the enzyme’s function. Studies of reaction
mechanisms have usually used biochemical and physicochemical methods such as spectroscopy. Using these techniques it is possible to determine the reaction mechanism by
characterizing the amino acids that directly participate in the
chemical reaction. However, the movements of side chains
that cooperate with the reaction are important for promoting
a smooth reaction. We have performed consecutive temporal
analyses of enzyme structures during the reactions to clarify
structure-based reaction mechanisms, including movements of
the side chains that do not directly contribute to the chemical
reactions.
‡ Research Fellow of the Japan Society for the Promotion of Science (JSPS).
58
doi:10.1107/S0909049510034989
Copper amine oxidases (EC 1.4.3.6) (CAOs) catalyze the
oxidative deamination of primary amines to their corresponding aldehydes with the concomitant production of
hydrogen peroxide and ammonia. In their active sites these
enzymes contain a copper ion and a redox-active organic
cofactor, 2,4,5-trihydroxyphenylalanine quinone (topaquinone, TPQox), which is a quinone cofactor that is covalently
linked to the polypeptide chain. TPQox is produced by the
post-translational modification of the conserved precursor
tyrosine residue in the presence of copper ion and molecular
oxygen.
We have studied the CAO phenylethylamine oxidase from
the Gram-positive bacterium Arthrobacter globiformis
(AGAO). AGAO is a homodimer comprised of two subunits,
each of which consists of 638 amino acid residues and has a
molecular weight of about 70 kDa. The mechanism of TPQox
biogenesis, i.e. the conversion of precursor apo-AGAO to
active holo-AGAO, has been identified by determining some
J. Synchrotron Rad. (2011). 18, 58–61
diffraction structural biology
Figure 1
The proposed mechanism of the catalytic reaction of AGAO.
of the major crystal structures of the biochemical reaction
intermediates of AGAO (Kim et al., 2002). A catalytic
mechanism which has also been proposed by using biochemical and physicochemical methods is shown in Fig. 1 (Mure et
al., 2002). According to this proposal the reaction is resolved
into two half-reactions. In the first reductive reaction the C5
carbonyl group of TPQox, its oxidized form, reacts with a
phenylethylamine (PEA) and forms the substrate Schiff-base
(TPQssb). The C1 proton from PEA is abstracted by a
conserved catalytic base, an aspartic acid residue, and forms
the product Schiff-base (TPQpsb). TPQpsb is hydrolyzed, which
gives rise to aminoresorcinol (TPQred), its reduced form, and
the release of the phenylacetaldehyde (HY1) product. TPQred
equilibrates with topasemiquinone (TPQsq) with simultaneously oxidizing the copper ion. In the subsequent oxidative
half-reaction, TPQsq is reoxidized to TPQox by molecular
oxygen, and ammonia and hydrogen peroxide are produced.
To identify the mechanism of the reductive half-reaction in
this catalytic reaction, the four reaction intermediates were
detected in the crystals and the structure at 15 min after the
onset of the reaction (R15) was determined at atomic resolution.
2. Materials and methods
2.1. Enzyme purification and crystallization
The precursor apo-AGAO was overexpressed in E. coli cells
and purified according to a previous procedure (Matsuzaki et
al., 1994). The purified solution of the precursor was dialyzed
against 50 mM copper sulfate buffer solution in order to
convert it to the active holo-AGAO, and was then concentrated to 10 mg ml1. Holo-AGAO was crystallized at 293 K
by microdialysis against 1.05 M potassium sodium tartrate
in 25 mM HEPES buffer, pH 6.8 (Kishishita et al., 2003), in
an anaerobic atmosphere in a glove box filled with N2 gas
( 99.9%). The dialysis buttons were transferred to a new
reservoir solution containing 45% (v/v) glycerol as a cryoprotectant (Wilce et al., 1997) for 1 day.
reaction intermediates. The crystals of the reaction intermediates were mounted on the loops and freeze-trapped by
injection into liquid nitrogen.
2.3. Single-crystal microspectroscopy
The crystals of the reaction intermediates were analyzed at
100 K by single-crystal microspectroscopy to determine the
stage of the catalytic reaction. The single-crystal microspectrophotometer system assembled by Dr Kawano, Riken
Harima, comprised a deuterium tungsten lamp, Cassegrain
mirrors, an optical fiber and a CCD-array spectrometer
(Ocean Optics, PC2000). Absorption spectra of the crystals
were recorded in the wavelength range 250–800 nm and
corrected by an air-blank reference. These spectra were
compared with spectra which were deconvoluted of chemical
species in solution (Chiu et al., 2006). From the results of
previous studies the wavelengths that gave the maximum
absorptions of the chemical species TPQox, TPQssb, TPQpsb,
TPQred and TPQsq were 480, 352, 425, 310 and 436/466 nm,
respectively.
2.4. Data collection and refinement
Diffraction data sets were collected at 100 K in a cold N2 gas
stream with synchrotron X-radiation ( = 1.000 Å) using an
ADSC CCD detector in the stations BL38B1 and BL44B2 at
SPring-8 (Hyogo, Japan). The data were processed and scaled
using HKL2000 (Otwinowski & Minor, 1997). The initial
phases of the data sets were determined by molecular replacement by holo-AGAO (Protein Data Bank accession code
2cfd) as a search model using MOLREP (Vagin & Teplyakov,
1997). The initial models were refined using REFMAC5
(Murshudov et al., 1997; Vagin et al., 2004). At the position of
residue 382, the models for TPQox/PEA, TPQssb, TPQpsb and
TPQsq/HY1 were built by using 2Fo Fc , Fo Fc and omit
maps. The refinement of the structure of R15 was completed
and the structure has been deposited in the RCSB Protein
Data Bank with the accession code 3AMO.
2.2. Trapping the reaction intermediates
3. Results and discussion
Plate-form crystals with approximate dimensions of 0.4 0.3 0.1 mm were selected. The crystals were scooped using a
thin nylon loop (diameter 0.3–0.4 mm) and then soaked in a
4 mM PEA solution for 1 to 120 min at 293 K in order to trap
3.1. Absorption spectra of the crystals
J. Synchrotron Rad. (2011). 18, 58–61
The UV/vis absorption spectra changes during the catalytic
reactions of AGAO in each crystal are shown in Fig. 2. Based
on the spectra of the crystals with deconvoluted absorptions,
Misumi Kataoka et al.
Copper amine oxidase
59
diffraction structural biology
Table 1
Statistics of data collection and crystallographic refinement of the crystal
of R15.
Figures in parentheses indicate the value for the highest-resolution shell (2.18–
2.10 Å).
Unit-cell dimensions
a (Å)
b (Å)
c (Å)
( )
Space group
Total number of observations
Total number unique reflections
Resolution (Å)
Completeness (%)
Rmerge (%)†
Multiplicity
I/(I)
Figure 2
Results of the UV/vis absorption spectra changes during catalytic
reaction of AGAO in each crystal with approximate dimensions of 0.4
0.3 0.1 mm. The crystals were soaked in PEA solution for 0, 1, 2, 5,
15, 30, 45, 60 and 120 min.
we found that the wavelength of the maximum absorption
spectrum of the crystal soaked in PEA for 2 min was close to
352 nm, consistent with that of TPQssb . Two peaks, 436 and
466 nm, arose with the passage of time; peaks at 60 min,
similar to 120 min, were 436 and 466 nm, which were consistent with those of TPQsq . These results suggested that AGAO
in crystals reacted with PEA during the time course from 2 to
60 min. In previous studies the reactive time in solution was
60 ms, whereas this time in crystals was 60 min. Therefore, the
catalytic reaction time of AGAO in crystals was prolonged by
as much as 6.0 104 times the reaction rate in solution
owing to the influence of the protein crystal field.
190.87
63.64
157.56
116.82
C2
631254
93540 (9425)
50.00–2.10 (2.18–2.10)
94.8 (96.1)
7.0 (36.3)
6.8 (5.9)
32.8 (6.5)
Refinements statistics
Rwork (%)‡
Rfree (%)§
Average B-factors (Å)
R.m.s. deviation from ideal values
Bond lengths (Å)
Bond angles ( )
18.9
24.5
28.1
0.021
1.9
† Rmerge = hi|Ih,i hIhi|/hiIh,i. ‡ Rwork = ||Fo| |Fc||/|Fo|. § Rfree = Rwork for
approximately 5% of the reflections that were excluded from the refinement.
and the reaction mechanism based on the intermediate
structures will be discussed elsewhere.
4. Conclusion
We have detected the reaction intermediates of AGAO in
crystals and confirmed these by two methods: single-crystal
3.2. Structural change in the substrate channel
The crystallographic statistics for R15 are shown in Table 1.
We determined four crystal structures for the catalytic reaction intermediates of AGAO. Fig. 3 shows the superposition of
chain B of R15 with the structure at 0 min (control). Based on
the Fo Fc omit map contoured at 2.4 the electron density
map of the TPQox position was connected to that of the PEA
position. We concluded that the product Schiff-base (TPQpsb)
was formed as a result of fitting models for TPQssb and TPQpsb
to the electron density map. This result almost corresponded
to the ratio of the time change of the amount of TPQpsb in the
crystal to that in solution from previous work by using the
mutant enzyme (Chiu et al., 2006). We also found conformational changes of the amino acid residues in the active site,
Phe105, and Leu3580 of a neighboring subunit that moved as a
cap for the substrate channel.
In this study we could reveal the structural changes through
an enzymatic reaction pathway not only for the active residues
and a cofactor but also for the residues that did not directly
participate in the chemical reaction. The detailed structures
60
Misumi Kataoka et al.
Copper amine oxidase
Figure 3
Superposed structures in the active site of crystals soaking in PEA
solution for 0 (control) (gray) and 15 min (magenta). Based on the Fo Fc omit map contoured at 2.4 s, it was revealed that the product Schiffbase (TPQpsb) was formed in this crystal. The substrate channel of AGAO
is shown by the (green) circle. The entrance of the substrate channel
(green arrow) was closed by Phe105 and Leu3580 of a neighboring subunit
that moved as a cap.
J. Synchrotron Rad. (2011). 18, 58–61
diffraction structural biology
microspectroscopy and X-ray crystal structure analysis. From
the measurements of UV/vis absorption spectra of the crystals
it was found that the two peaks for TPQsq arose during the
time course of the experiment, but the spectra for the reaction
intermediates could not be observed. However, using X-ray
crystallography we succeeded in trapping and determining the
reaction intermediates. Our results suggest that the reaction
intermediates came to exist as a mixture, as the rates of
reactions that were controlled in the crystals by substrate
diffusion were not completely synchronized between the
surface and the core, and because the reaction rate was
dependent on each elementary reaction. In addition, the
radiating beam positions for both single-crystal microspectroscopy and X-ray did not match entirely. Therefore, it is
suggested that the absorption peak of a chemical species that
had a small absorption was hidden by that of a chemical
species that had a large absorption. If these experiments are
carried out by irradiating the entire region using a smaller
crystal or a microcrystal, the results from single-crystal
microspectroscopy would correspond to X-ray crystal structure analysis. However, the detection of a significant electron
density map for a reaction intermediate suggests that the
reaction intermediate accumulates during that time. In
conclusion, the results obtained here provide quantitatively
sufficient information to trace the structural changes of a
reaction.
The chemical reaction rate in a protein crystal generally
becomes slow owing to limited protein motion under the
influence of a protein crystal field. In this study the rate of the
catalytic reaction of AGAO in crystals was 6.0 104 times
slower than that in solution. The reactions after soaking and
trapping intermediates using cryogenic temperatures could
become a general method for determining the intermediate
structures of other proteins.
J. Synchrotron Rad. (2011). 18, 58–61
This work was supported by a Grant-in-Aid for Research
Fellow of the Japan Society for the Promotion of Science
(JSPS), and in part by a Grant-in-Aid for Physical Science
Center for Biomolecular Systems Research. We thank Drs
Yoshikazu Kawano, Takaaki Hikima, Masaki Yamamoto
(RIKEN Harima Institute, Japan) and Takashi Kumasaka
(JASRI, Japan) for the single-crystal microspectrophotometer
measurement. We appreciate useful discussions with Dr
Keiichi Hosokawa
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